PTEN, a dual-specificity phosphatase, exerts its tumor-suppressive effects through the inhibition of cell cycle progression and cell immigration, therefore could be an important candidate for tumor-suppression. As study on prokaryotic expression of PTEN and its anti-tumor functions has not been reported, the present study aims at an efficient expression of functional PTEN in Escherichia coli and the investigation of its tumor-suppression activity. PTEN cDNA cloned in our lab previously was recombined into prokaryotic expression vector pET-44a(+) to construct pET-PTEN (pEP) and pET-Nus-PTEN (pENP). PTEN was fused with 6 x His tag in pEP, and with Nus in pENP, which could be useful for a stable and soluble expression. The recombinant vectors were transformed into both BL21 (DE3) (BL) and Rosetta-gami (DE3) pLysS (RG). The former is a normal expression host while the latter is optimized for expression of eukaryotic genes and folding of target proteins. On the induction of 0.5mmol/L IPTG, 55kD and 118kD specific protein bands were observed, corresponding to His-PTEN and Nus-PTEN fusion proteins, respectively. Western blot analysis showed the recombinant fusion proteins could react with PTEN polyclonal antibody. The recombinant HTEN was expressed both in soluble fraction and inclusion body. Higher expression levels of recombinant PTEN were obtained in BL (His-PTEN: 10.3%; NusA-PTEN: 18.7%), whereas the higher percentages of soluble recombinant proteins were observed in RG (His-PTEN: 4.7%; Nus-PTEN: 6.6%). The obtained recombinant fusion proteins were purified by affinity chromatography and were showed to be homogeneous in SDS-PAGE. In tumor-suppression experiments, His-PTEN was proved to have significant inhibition on growth of mice solid tumor with an inhibitory ratio of 58.76%, and on the proliferation of DU-145 tumor cells with an inhibitory ratio of 46.16%. The cell cycle progression of DU-145 tumor cells was also arrested from G0/G1 to S phase. His-PTEN from RG was proved to have significantly higher tumor-suppression activity than that from BL, indicating that there may be some advantages for eukaryotic genes to be expressed in the former host. This is the report of functional recombinant PTEN expressed in Escherichia coli. Purified His-PTEN was used for immunizing Kunming mice, and ascitic polyclonal antibodies raised against His-PTEN were generated using sarcoma 180 cells. At 1:2000 dilution, the antibodies could interact with PTEN by western blot. The present study has laid a foundation for application of PTEN in cancer therapy.
Animals ; Antibodies, Neoplasm ; biosynthesis ; immunology ; Antineoplastic Agents ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Humans ; Liver Neoplasms, Experimental ; therapy ; Mice ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo investigate the morphological changes after chemically extracted acellular nerve allografts transplant.

METHODSSeventy-two rabbits were divided into four groups. Acellular allografts of facial nerve were used in experimental group, and facial nerve autografts, acellular peroneal nerve allografts and peroneal nerve autografts respectively used in three control groups. The morphological changes after transplant were evaluated by modified trichrome staining, immunohistological staining and transmission electron microscope.

RESULTSThe two facial nerve grafts showed numerous regenerated nerve fibers, vessels and as well as a spindle schwann cells arranged longitudinally. No significant difference was observed in the fiber number and myelin thickness between the two groups,while the two peroneal nerve groups showed poor regeneration 6 months after operation.

CONCLUSIONSThe facial nerve allografts showed more neurite regeneration six months after transplant, and the regenerated axons passed through the distal stoma and there were well revascularized and proliferated schwann cells in the grafts.

Animals ; Disease Models, Animal ; Facial Nerve ; pathology ; transplantation ; Facial Nerve Injuries ; pathology ; surgery ; Nerve Regeneration ; Rabbits ; Transplantation, Homologous

OBJECTIVETo detect the expression of D-E-A-D-box polypeptide 41 (DDX41) in human dental pulp tissues and cells.

METHODSThe mRNA and protein expressions of DDX41 in human dental pulp cells were detected by RT-PCR and immunocytochemistry, and the expression of DDX41 in human dental pulp tissues was investigated by immunohistochemistry.

RESULTSStrong expressions of DDX41 mRNA and protein were detected in dental pulp cells. In dental pulp tissues, DDX41 was expressed in the cytoplasm and nucleus of odontoblasts.

CONCLUSIONDDX41/STING-dependent TBK1-IRF3-IFN-β signaling pathway may play a role in innate immune responses of the dental pulp to caries and pulpitis.

Cell Nucleus ; metabolism ; Cells, Cultured ; Cytoplasm ; metabolism ; DEAD-box RNA Helicases ; metabolism ; Dental Pulp ; metabolism ; Humans ; Immunohistochemistry ; Odontoblasts ; metabolism ; RNA, Messenger ; Signal Transduction

OBJECTIVETo evaluate the effect of SMU.2055 gene on acid resistance of Streptococcus mutans.

METHODSA SMU.2055-dificient mutant strain of S. mutans was constructed using homologous recombination technique. The growth of the wild-type and mutant strains was monitored in both normal and acidic conditions. The lethal pH level, glycolysis, proton permeability, cell permeability and biofilm formation of the two strains were compared.

RESULTSPCR and sequence analyses verified the successful construction of the SMU.2055-dificient mutant strain. The growth and biofilm formation capacity of the mutant strain were obviously lowered in both normal and acidic conditions. The mutant strain also showed increased lethal pH level, proton permeability, and cell permeability with impaired H-ATPase activity in acidic conditions, but its minimum glycolytic pH remained unaffected.

CONCLUSIONThe SMU.2055-deficient S. mutans mutant exhibits a lowered acid resistance, which affects the growth, lethal pH, proton permeability, H-ATPase activity, cell permeability and biofilm formation but not the minimum glycolytic pH of the mutant strain.

OBJECTIVE: To investigate ultrasound and MRI features of malignant fibrous histiocytoma (MFH) of soft tissue. METHODS: Ultrasound, MRI images and pathological data of 12 patients with malignant fibrous histiocytoma in soft tissue confirmed by operation and pathology were analyzed from January 2012 to August 2018, inlcuding 7 males and 5 females, aged from 36 to 69 years old with an average age of 53 years old; the courses of disease ranged from 4 to 49 months with an average of 28 months. Clinical manifestations were soft tissue masses and pain in the affected limbs. Ultrasound, MRI and contrast-enhanced examination were performed before operation. The lesions, morphology, echo/signal characteristics, color flow signals and enhancement features were observed and compared with pathology. RESULTS: In 12 patients with MFH, 9 patients were primary lesions and 3 patients were recurrent lesions after operation. There were 7 cases of bilateral thighs, 2 cases of calves, 1 case of upper arm, 1 case of buttocks and 1 case of posterior peritoneum. The size ranged from 5.1 to 17.1 cm with an average of 8.7 cm. Ultrasound feature showed lobulated or agglomerate, and focused on low echo; 5 cases had capsule and with clear border; 7 cases were unclear boundary with surrounding tissues; and 6 cases with irregular echo-free. The blood flow signals were around the CDFI, and the internal blood flow signals were different. MRI feature showed lobulated, agglomerate or irregular shape, T1WI showed slightly lower signal or equal signal, T2WI showed high signal and DWI signal increased. Six patients manifested mixed signal inside, 7 patients manifested low signal separation inside, 5 patients with false envelope, and 9 patients manifested infiltration and growth with peripheral edema. T1WI showed uneven strengthening after enhancement. Immunohistochemical expression of Vim, CD68 were positive. CONCLUSIONS The age, location and imaging features of soft tissue MFH are characteristic. The diagnosis of MFH should be considered when irregular mass occurred in soft tissues of limbs at middle-aged and old people. Echo and signal are homogeneous or mixed. Separation, necrosis and cystic degeneration could be seen in the mass. When the blood flow signals are abundant and solid components are obviously enhanced, the diagnosis of MFH should be considered.
Adult ; Aged ; Edema ; Extremities ; Female ; Histiocytoma, Malignant Fibrous ; diagnostic imaging ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Ultrasonography

OBJECTIVETo investigate the association between single nucleotide polymorphisms (SNPs) in DNA methyltransferase 1 (DNMT1) (rs12984523, rs16999593, and rs2228612) and noise-induced hearing loss (NIHL) in Chinese Han population.

METHODSThis case-control study consisted of 188 cases (case group) and 300 controls (control group) in the same working position, who were matched for age and gender. The cases had a binaural average high-frequency hearing threshold not less than 40 dB, and the controls had a binaural average high-frequency hearing threshold less than 40 dB. The genotypes at the three SNPs were determined by TaqMan probe.

RESULTSTT genotype at DNMT1 rs2228612 is a risk factor for NIHL (adjusted OR = 1.69, 95% CI: 1.14-2.52).

CONCLUSIONThe study of Chinese Han population suggested that DNMT1 rs2228612 is associated with susceptibility to NIHL.

Adult ; Asian Continental Ancestry Group ; genetics ; Auditory Threshold ; Case-Control Studies ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Female ; Genetic Predisposition to Disease ; Genotype ; Haplotypes ; Hearing Loss, Noise-Induced ; genetics ; Humans ; Male ; Middle Aged ; Noise, Occupational ; Polymorphism, Single Nucleotide ; Risk Factors

Primate placentation involves a series of cell proliferation, immigration and apoptosis which account for the progressive tissue remodelling at the implantation site. p53 is an important proto-oncogene involved in the regulation of cell-cycle and apoptosis. To study the effect of RU486 on apoptosis and expression of p53 at the fetal-maternal interface, the location of apoptotic cells and expression of p53 were examined using in situ 3'-end labeling method, immunohistochemistry and Western blot assay at the fetal-maternal interface of normal and RU486 treated rhesus monkey. Western blot analysis showed the specificity of the anti-human antibody used with the monkey tissue. In the placental villi, the apoptotic nuclei were observed mainly in the syncytiotrophoblast and part of the cytotrophoblast within the cell column; p53 protein was detected mainly in the cytotrophoblast. In the endometrium, positive signals for apoptosis and p53 were detected in some stromal cells. After two days of mifepristone treatment, the apoptotic cells increased significantly in both placental villi and endometrium. In the villi, the increased apoptotic nuclei were mainly localized to the cytotrophoblast. At the same time, p53-positive nuclei also increased in both villous cytotrophoblast cells and endometrial stromal cells, after the treatment of RU486. These results suggest that apoptosis and expression of p53 are essential in regulating trophoblastic homeostasis by controlling its proliferation in normal placenta, whereas the up-regulation of p53 protein may play an important role in apoptosis that happens at the fetal-maternal interface induced by RU486.
Abortifacient Agents, Steroidal ; pharmacology ; Animals ; Apoptosis ; drug effects ; Chorionic Villi ; pathology ; Female ; Macaca mulatta ; Mifepristone ; pharmacology ; Placentation ; drug effects ; physiology ; Pregnancy ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo obtain stable primary cultures of human malignant meningioma cells and establish an intracranial in-situ tumor model in nude mice.

METHODSTen surgical specimens of highly suspected malignant meningioma were obtained with postoperative pathological confirmation. Primary malignant meningioma cells were cultured from the tissues using a modified method and passaged. After identification with cell immunofluorescence, the cultured cells were inoculated into the right parietal lobe of 6 nude mice using stereotaxic apparatus and also transplanted subcutaneously in another 6 nude mice. The nude mice were executed after 6 weeks, and HE staining and immunohistochmistry were used to detect tumor growth and the invasion of the adjacent brain tissues.

RESULTSThe primary malignant meningioma cells were cultured successfully, and postoperative pathology reported anaplastic malignant meningioma. Cell immunofluorescence revealed positivity for vimentin and EMA in the cells, which showed a S-shaped growth curve in culture. Flow cytometry revealed a cell percentage in the Q3 area of (95.99∓2.58)%. Six weeks after transplantation, tumor nodules occurred in the subcutaneous tumor group, and the nude mice bearing the in situ tumor showed obvious body weight loss. The xenografts in both groups contained a mean of (36∓5.35)% cells expressing Ki-67, and the intracranial in situ tumor showed obvious invasion of the adjacent peripheral brain tissues.

CONCLUSIONWe obtained stable primary cultures of malignant meningioma cells and successfully established a nude mouse model bearing in situ human malignant meningioma.

Adolescent ; Adult ; Aged ; Female ; Humans ; Laparoscopy ; Male ; Middle Aged ; Splenectomy ; methods

OBJECTIVETo study the changes of expression of Survivin mRNA, BCRP mRNA and HER-2 mRNA in breast cancer after TE regimen neoadjuvant chemotherapy, and to find biological markers to predict the efficiency of TE regimen neoadjuvant chemotherapy.

METHODSThe gene expressions were detected by RT-PCR from 56 breast cancer patients before and after TE regimen neoadjuvant chemotherapy (docetaxel and epirubicin). The relationships between these gene expressions and chemotherapy responses were analyzed.

RESULTSThe overall response rate to neoadjuvant chemotherapy was 71.4%, including 8.9% (5/56) with complete response and 62.5% (35/56) with partial response. Pathological complete response was found in 4 cases (7.1%). Stable disease and progression of disease were 23.2% (13/56) and 5.4% (3/56), respectively. The expression of Survivin mRNA after neoadjuvant chemotherapy was 35.7% (20/56), significantly lower than 60.7% (34/56) before neoadjuvant chemotherapy (P = 0.008). The expression of BCRP mRNA after neoadjuvant chemotherapy was 19.6%, significantly lower than 37.5% before neoadjuvant chemotherapy (P = 0.036). The positive rate of HER-2 mRNA expression was 41.1% before the chemotherapy, and reduced to 21.4% after the chemotherapy (P = 0.025). The effective rates of the single positive expression of Survivin mRNA or BCRP mRNA were both lower than that of negative expression (P < 0.05). The level of HER-2 mRNA expression alone was not significantly associated with the effective rate of chemotherapy (P = 0.144). When the expression of all Survivin mRNA, BCRP mRNA and HER-2 mRNA were negative, the effective rate of neoadjuvant chemotherapy was higher than that in patients with positive expression (P = 0.003). The level of Survivin mRNA expression was not significantly associated with BCRP mRNA and HER-2 mRNA (P > 0.05).

CONCLUSIONThe expression of Survivin in combination with BCRP and HER-2 is associated with clinical response to TE neoadjuvant chemotherapy in breast cancer, and can be used as predictive biomarkers for chemosensitivity of TE regimen neoadjuvant chemotherapy for breast cancer.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Breast Neoplasms ; drug therapy ; metabolism ; surgery ; Carcinoma, Ductal, Breast ; drug therapy ; metabolism ; surgery ; Carcinoma, Lobular ; drug therapy ; metabolism ; surgery ; Disease Progression ; Epirubicin ; administration & dosage ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Mastectomy, Radical ; methods ; Middle Aged ; Neoadjuvant Therapy ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptor, ErbB-2 ; genetics ; metabolism ; Remission Induction ; Taxoids ; administration & dosage